Ribosomal RNA depurination prevents the unfolded protein response through translation inhibition

Many newly translated proteins are targeted to the endoplasmic reticulum (ER) for folding, processing and transport to extracellular spaces. The unfolded protein response (UPR), mediated by the ER stress sensor Ire1 that splices mRNA of transcription factor Hac1, is activated when unfolded proteins accumulate. However, it remains unclear how ribosome integrity, through its effects on translation, controls UPR. To address this question, we expressed pokeweed antiviral protein (PAP), an rRNA N-glycosylase that depurinates 25S rRNA in Saccharomyces cerevisiae. We compared the effect of PAP with an enzymatically inactive mutant PAPx, as both proteins are targeted to the ER during translation, but only one damages ribosomes. RNA sequencing across a six-hour time course revealed enrichment of the ER protein processing pathway and selective upregulation of UPR-related genes in PAPx-expressing cells, but not in PAP-expressing cells. PAPx expression also increased HAC1 splicing, whereas PAP did not, even after tunicamycin treatment. Additionally, Ire1 remained inactive, as shown by lack of Ire1-2xGFP oligomerization viewed by confocal microscopy. Absence of UPR activation in PAP-expressing cells was correlated with reduced azidohomoalanine (methionine analog) incorporation into membrane-associated proteins, consistent with reduced protein flux into the ER. Our findings support a new model describing how rRNA depurination inhibits UPR upstream of Ire1 activation by reducing translational capacity, which maintains Ire1 in an inactive state. Therefore, ribosome integrity influences UPR activation by controlling translation and membrane-associated protein synthesis.